Method for using unequal primer concentrations for generating nucleic acid amplification products

ABSTRACT

The method provided herein is a method for detecting a target sequence in a test sample. Generally, the method comprises forming a reaction mixture comprising a test sample, amplification reagents, a first primer, and a second primer wherein the concentration of the first primer in the reaction mixture is 15% to 250% percent greater than the concentration of the second primer. The target sequence is amplified according to any amplification protocol that employs the primer sequences to generate copies of the target sequence that include a product from the first and second primers. A probe is hybridized to the amplification product from the first primer to form a hybrid complex; and the hybrid complex is detected as an indication of the presence of the target sequence in the test sample.

This application is a continuation of U.S. application Ser. No.09/079,675, filed May 15, 1998 now U.S. Pat. No. 6,391,544.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to nucleic acid amplification reactionsand in particular relates to amplification reactions that employ a pairof primer sequences to generate copies of a target sequence.

BACKGROUND OF THE INVENTION

Nucleic acid amplification reactions are well known and are employed toincrease the concentration of a target nucleic acid in a test sample.The “target nucleic acid” typically is present in a sample in lowconcentrations and therefore cannot easily be detected withoutamplifying it to increase the concentration of the target sequence inthe sample. The polymerase chain reaction (PCR) is one nucleic acidamplification reaction commonly employed for purposes of amplifying atarget nucleic acid sequence.

According to the principles of PCR, “primer sequences” are used to primesynthesis of copies of the target sequence. Specifically, underappropriate conditions, primer sequences hybridize to opposite strandsof a double stranded nucleic acid sequence such that the primers flankthe target sequence. Once hybridized, the primers are extended usingenzymes such as, for example, DNA polymerase which extend the primersequences to thereby generate copies of the target sequence. Additionalcopies of the target sequence are generated by cycling the above stepsof (i) hybridizing and extending the primer sequences and (ii)dissociating the extended primer sequences (or copies of the targetsequence) so that additional primers can hybridize to the originaltarget, as well as copies of the target sequence. Hence, multiple copiesof the target sequence are generated.

Once amplified, copies of the target sequence can be detected todetermine if the target sequence originally was present in the testsample. Of course, if the target sequence was not present, amplificationshould not occur and the target sequence should not be detected. In anyevent, amplified target sequences are typically detected using labels.Labels are moieties that have a detectable property and can beincorporated into the copies of the target sequence. Labels typicallyare incorporated into the amplified target sequences by attaching thelabels to primer sequences that are then incorporated into theamplification product as specified above. Alternatively, for example,extension products can be labeled by incorporating labeled nucleotidesinto such products during primer extension. The presence of the targetsequence in the test sample can then be determined by detecting thelabeled amplification product.

Amplified target sequences also can be detected using labeled probesthat hybridize to a strand or both strands of an amplified targetsequence. However, it is sometimes desirable to employ a probe thathybridizes to only one strand of a double stranded amplificationproduct. The effect of such a detection scheme, at least as it appliesto a double stranded target sequence, is that a single strand ofamplified target sequence is detected to determine the presence of thetarget sequence in the test sample. However, detecting a single strandof an amplification product can be inefficient insofar as the signalplateaus and sometimes drops (or hooks) as the number of targetsequences originally present in the test sample increases. Alleviatingthe “hooking” or “plateauing” phenomenon and providing a linear signalover a broader range of target sequence concentrations would bebeneficial, especially for amplification based assays designed toquantify the amount of a target sequence in a test sample.

It would be expected that substantially increasing the concentration ofone primer over the other would alleviate this problem by generatingmore of the sequence that is detected. Indeed, U.S. Pat. No. 5,066,584describes a method for preferentially generating one strand of a doublestranded target sequence by vastly increasing the concentration of oneprimer. However, this requires excess reagents and therefore excesscosts associated with preferentially producing one of two singlestrands. Additionally, substantially increasing primer concentrationsmay increase the chances of non-specific priming and thereforeamplification of non-target sequences. Moreover, many times, competingnon-specific reactions will interfere with the efficient amplificationof the sequence of interest. Therefore, it may be expected thatsubstantially increasing the concentration of one primer over the otherprimer may present problems in amplification assays designed to be ofhigh sensitivity (i.e. designed to detect low numbers of a sequence ofinterest).

SUMMARY OF THE INVENTION

The present invention provides a method of detecting a target sequencein a test sample. The method comprises the steps of: (a) forming areaction mixture comprising a test sample, amplification reagents, afirst primer, and a second primer such that the concentration of thefirst primer in the reaction mixture is 15% to 250% percent greater thanthe concentration of the second primer; (b) amplifying the targetsequence to generate copies of the target sequence comprising anamplification product from the first and second primers; (c) hybridizinga probe to the amplification product from the first primer to form ahybrid complex; and (d) detecting the hybrid complex as an indication ofthe presence of the target sequence in the test sample. Preferably, thehybrid complex is detected using labels that can either be directlydetectable or indirectly detectable.

Also provided is an improved method for amplifying and detecting atarget nucleic acid sequence in a test sample comprising the steps of:(a) forming an amplification mixture comprising a test sample, a firstand a second primer sequence, and amplification reagents, (b) amplifyingthe target sequence to generate copies of the target sequence comprisingan amplification product from the first and second primers; and (c)detecting the copies of the target sequence as an indication of thepresence of the nucleic sequence in the test sample; wherein theimprovement comprises providing the first primer sequence in 15% to 250%excess over the second primer and wherein a probe is hybridized to theamplification product from the first primer to form a hybrid complex andthe hybrid complex is detected as an indication of the presence of thenucleic acid sequence in the test sample.

Kits for performing the methods of the invention are also provided.

DETAILED DESCRIPTION OF THE INVENTION

Under appropriate conditions, a primer pair will generate copies of atarget sequence in the form of a double stranded amplification product.Unfortunately, however, when a single strand of a double strandedamplification product is detected with a probe, the resulting signal canplateau, or even hook, as the concentration of the original targetsequence increases. To a certain extent, this phenomenon iscounterintuitive since increasing the concentration of the originaltarget sequence should yield a greater concentration of end product, andtherefore, a greater signal should be detected. However, as mentionedabove, the resulting signal can plateau. While not wishing to be boundby theory, the hooking effect may be attributable to the presence ofhigher concentrations of longer product strands driving product strandre-annealing to the exclusion of probe/target strand annealing.Applicants have surprisingly and unexpectedly discovered that theplateauing or hooking phenomenon could be alleviated by increasing theconcentration of one primer so that it is slightly higher than theconcentration of the other primer.

The method provided herein can be applied to any amplification reactionwhere a pair of primer sequences is employed to generate double strandedamplification products and only one strand of the double strandedproducts is detected. The method comprises a step where an amplificationmixture is formed. The amplification mixture generally will comprise (i)a test sample, (ii) amplification reagents and (iii) a first and secondprimer (collectively referred to as a “primer pair”). As used herein,the term “test sample” means anything suspected of containing a targetsequence. The test sample is or can be derived from any biologicalsource, such as for example, blood, ocular lens fluid, cerebral spinalfluid, milk, ascites fluid, synovial fluid, peritoneal fluid, amnioticfluid, tissue, fermentation broths, cell cultures and the like. The testsample can be used directly as obtained from the source or following apre-treatment to modify the character of the sample. Thus, the testsample can be pre-treated prior to use by, for example, preparing plasmafrom blood, disrupting cells or viral particles, preparing liquids fromsolid materials, diluting viscous fluids, filtering liquids, distillingliquids, concentrating liquids, inactivating interfering components,adding reagents, purifying nucleic acids, and the like. The “targetsequence” that may be present in the test sample is a nucleic acidsequence that is amplified, detected, or amplified and detected.Additionally, while the term target sequence is sometimes referred to assingle stranded, those skilled in the art will recognize that the targetsequence may actually be double stranded.

The phrase “amplification reaction reagents” as used herein meansreagents which are well known for their use in nucleic acidamplification reactions and may include but are not limited to: anenzyme or enzymes separately or individually having DNA polymeraseand/or reverse transcriptase activity; enzyme cofactors such asmagnesium or manganese; salts; nicotinamide adenine dinucleotide (NAD);deoxynucleoside triphosphates (dNTPs) such as, for example,deoxyadenosine triphosphate, deoxyguanosine triphosphate, deoxycytodinetriphosphate and thymidine triphosphate; and an appropriate buffer.

The first and second primers typically are nucleic acid sequences,usually DNA or RNA. The length of the primers is not critical but primersequences are usually about 10 to about 100 nucleotides long, preferablyfrom about 15–35 nucleotides long, and have a defined base sequencesuitable for hybridizing to the desired target sequence. Primer pairsusually are selected such that they flank the target sequence as is wellknown in the art. Additionally, the first primer is added to theamplification mixture such that its concentration is between 15% to 250%greater, and preferably 20% to 150% greater, than the concentration ofthe second primer. Of course, the first primer can be employed atconcentrations of 400%, 500% and up to and more than 1000% greater thanthe concentration of the second primer, but as the concentration of oneprimer over the other increases, reagent costs and/or non-specificpriming can become a limiting factor. In any event, upon hybridizationof a primer to a target sequence, the primer is extended to generate acomplement of the sequence to which the primer is hybridized.

Primer sequences can be from natural or synthetic sources and canroutinely be synthesized using a variety of techniques currentlyavailable. For example, primers can be synthesized using conventionalnucleotide phosphoramidite chemistry and instruments available fromPerkin Elmer/Applied Biosystems, Div., (Foster City, Calif.) orPerceptive Biosystems, Inc., (Framingham, Mass.). If desired, a primercan be labeled using methodologies well known in the art such asdescribed in U.S. Pat. Nos. 5,464,746; 5,424,414; and 4,948,882.

After the amplification mixture is formed, the target nucleic acid isamplified by subjecting reaction mixture to “amplification conditions”which are conditions that promote amplification of the target sequence.Amplification conditions are well known to those skilled in the art andgenerally comprise conditions that promote dissociation of a doublestranded target sequences, annealing of the primer sequences to thesingle strands of the target sequence, and extension of the primersequences to thereby form copies of the target sequences. The copies ofthe target sequence are then dissociated from the target and additionalprimer sequences are annealed to both the original target sequence andcopies of the target sequence to thereby start a new round ofamplification of the target sequence. Such amplification conditions arewell known and have been described in U.S. Pat. Nos. 4,683,202 and4,683,195 both of which are herein incorporated by reference. Thermalcycling is a preferred and well known method for producing amplificationconditions. The number of times an amplification mixture is cycled is amatter of choice for one skilled in the art and typically, a reactionmixture is cycled between 2 and 100 times and more typically between 20and 40 times.

After cycling, multiple copies of the target sequence may be present.The sequence(s) generated by the first primer is the sequence that isdetected to indicate the presence of the target sequence in the testsample and this sequence synthesized by the first primer is variouslyreferred to herein as the “primary sequence”. Any method for detecting asingle strand of a double stranded amplification product can be employedaccording to the present invention. For example, sequencing, gelelectrophoresis, gel shift assays, solution hybridization assays,“TaqMan”like assays, and similar formats can be employed to detect theprimary sequence.

According to a preferred detection embodiment, a hybridization probe isemployed to detect the primary sequence, particularly when the probe isat relatively low concentrations. Probe sequences hybridize to theprimary sequence to form a hybrid complex. Preferably, probes hybridizeto the primary sequence in a region that is internal with respect to theprimers. Formation of a hybrid complex between the primary sequence andprobe can be accomplished by placing any double stranded targetsequences under dissociation conditions followed by placing anyresultant single stranded sequences under hybridization conditions inthe presence of a probe. The phrase “dissociation conditions” is definedgenerally as conditions which promote dissociation of double strandednucleic acid to the single stranded form. These conditions can includehigh temperature and/or low ionic strength. The phrase “hybridizationconditions” is defined generally as conditions which promote nucleationand annealing of complementary nucleic acid sequences. It is well knownin the art that such annealing and hybridization is dependent in arather predictable manner on several parameters, including temperature,ionic strength, sequence length and G:C content of sequences. For anygiven set of sequences, melt temperature, or Tm, can be estimated by anyof several known methods. Typically hybridization conditions includetemperatures which are slightly below the melt temperature of given setof nucleic acid sequences. Ionic strength or “salt” concentration alsoimpacts the melt temperature, since small cations tend to stabilize theformation of duplexes by shielding the negative charge on thephosphodiester backbone. Typical salt concentrations depend on thenature and valence of the cation but are readily understood by thoseskilled in the art. Similarly, high G:C content and increased sequencelengths are also known to stabilize duplex formation because G:Cpairings involve 3 hydrogen bonds where A:T pairs have just two, andbecause longer sequences have more hydrogen bonds holding the strandstogether. Thus, a high G:C content and longer sequence length impactwhat “hybridization conditions” will encompass. Based upon the above,determining the proper “hybridization conditions” for a particular setof nucleic acid sequences is well within the ordinary skill in the art.U.S. patent application Ser. No. 08/514,704, filed Aug. 14, 1995,exemplifies a method of detecting amplified target sequences with aprobe.

Probes are also nucleic acid sequences or nucleic acid analog sequencessuch as, for example, DNA, RNA, peptide nucleic acids, morpholinonucleic acids, that can be synthesized and labeled in the same mannerthat primer sequences are synthesized and labeled, as specified above.Selection of labels employed on a labeled primer or probe is a matter ofchoice for those skilled in the art and the term “label” as used hereinrefers to a molecule or moiety having a property or characteristic whichis capable of detection. A label can be directly detectable, as with,for example, radioisotopes, fluorophores, chemiluminophores, enzymes,colloidal particles, fluorescent microparticles, FRET pairs, and thelike. Alternatively, a label may be indirectly detectable, as with, forexample, specific binding members. It will be understood that directlydetectable labels may require additional components such as, forexample, substrates, triggering reagents, light, and the like to enabledetection of the label. When indirect labels are used for detection,they are typically used in combination with a conjugate as will bediscussed further below.

Probes can be employed in a variety of ways, known in the art, to detectthe primary sequence. For example, the probe, primer or probe and primercan be labeled and/or immobilized to solid suppert materials to detectthe presence of the primary sequence. Capture reahents also can beemployed to aid in detecting a primary sequence. A “capture reagent” asused herein means a specific binding menber attached to a solid supportmaterial. “Specific binding member” as used herein, means a ember of aspecific biding pair, i.e. two different molecules where one of themolecules through, for example, chemical or physical means specificallybinds to the other molecule. In addition to antigen and antibodyspecific binding pairs, other specific binding pairs include, but arenot intended to be limited to avidin and biotin; complementarynucleotide sequences; haptens and antibodies specific for haptens suchas carbazole and adamantane described in U.S. Pat. Nos. 5,424,414 and5,464,746, respectively; and the like. A “solid support material”,refers to any material which is insoluble, or can be made insoluble by asubsequent reaction. Solid support materials thus can be latex, plastic,derivatives plastic, magnetic or non-magnetic metal , glass, silicon orthe like. A vast array of solid support material configurations are alsowell known and include, but are not intended to be limited to, surfacesof rest tubes, microtiter wells, sheets, beads, microparticles, chipsand other configurations well know to those skilled in the art.

According to one embodiment for detecting a primary sequence using aprobe, the probe can be immobilized to a solid support material to forma capture reagent. The primary sequence can be contacted with theso-formed capture reagent under hybridization conditions to form ahybrid complex and thereby capture the primary sequence and, if desired,separate it from other amplification reactants and products. A signalfrom a label attached to the primary sequence can then be detected as anindication of the presence of the primary sequence on the capturereagent and therefore in the test sample.

As a further alternative, the first primer and probe can be labeled andthe primary sequence can be separated and detected using such labels.For example, both labels of such a configuration can be specific bindingmembers. Hence upon formation of a hybrid complex, the complex will bebi-labeled. One label can bind to a specific binding member on a capturereagent that permits separation of the hybrid complex and the otherlabel can be used to bind a conjugate which can be employed to detectthe presence of the hybrid complex on the capture reagent. The term“conjugate” as used herein means a specific binding member that has beenattached or coupled to a directly detectable label. Coupling chemistriesfor synthesizing a conjugate are well known in the art and can include,for example, any chemical means and/or physical means that does notdestroy the specific binding property of the specific binding member orthe detectable property of the label.

EXAMPLES

The following examples demonstrate detection of HIV nucleic acid usingthe DNA oligomer primers and probes herein provided. These DNA primersand probes are identified as SEQUENCE ID NO. 2, SEQUENCE ID NO. 3 andSEQUENCE ID NO. 4 and are specific for a region in the pol gene of HIV.A portion of a representative pol sequence from HIV-1 (subtype B, strainMN) is designated herein as SEQ ID NO. 1. These primers and probes areconsensus sequences derived from analysis of the pol region of 31 HIV-1isolates, representing subtypes A through F and O of HIV-1.

In the following examples, SEQ ID NO. 2 and SEQ ID NO. 3 are used asconsensus amplification primers specific for the pol region of HIV-1.SEQ ID NO. 4 is used as a consensus internal hybridization probe for theHIV-1 pol amplification product.

Example 1 Preparation of HIV Primers and Probes

A. HIV Primers. Consensus primers were designed to detect the HIV poltarget sequence of all known HIV-1 subtypes by oligonucleotidehybridization PCR. These primers were SEQ ID NO. 2 and SEQ ID NO. 3.Primer sequences were synthesized using standard oligonucleotidesynthesis methodology, and SEQ ID NO. 3 was haptenated with carbazole atthe 5′ end using standard cyanoethyl phosphoramidite coupling chemistryas described in U.S. Pat. No. 5,424,414.B. HIV Probes. The consensus probe, designated SEQ ID NO. 4, wasdesigned to hybridize with the amplified HIV pol target sequences byoligonucleotide hybridization. The probe sequence was synthesized usingstandard oligonucleotide synthesis methodology and haptenated with 2adamantanes at the 5′ end using standard cyanoethyl phosphoramiditecoupling chemistry as described in U.S. Pat. No. 5,464,746 and blockedwith phosphate at the 3′ end.

Example 2 Detection of HIV Varying the Unlabeled Primer Concentration

HIV RNA was isolated from a known quantity of virions (AdvancedBiotechnologies Inc., Columbia, Md.) using RNAzol B RNA IsolationSolvent (Tel-Test, Inc., Friendswood, Tex.), extracted withchloroform/isopropanol and precipitated with ethanol. The pellet wasresuspended in RNase-free water (5′-3′, Boulder Colo.). Ten-folddilutions of this HIV RNA were then prepared at concentrations of 10⁶ to10¹ RNA molecules/25 μl using a diluent containing 2 ng/μl of ribosomalRNA (rRNA; Boehringer-Mannheim, Indianapolis Ind.).

Dilutions of the HIV RNA (excluding 10⁴) were reverse transcribed, PCRamplified and detected using SEQ ID NOs. 2 and 3 as primers with SEQ IDNO. 4 as the HIV probe. RT-PCR was performed using 1×EZ Buffer, 2.5 mMmanganese chloride, dNTPs (dATP, dGTP, dTTP and dCTP) present at a finalconcentration of 0.15 mM each, and recombinant Thermus thermophiluspolymerase at a concentration of 5 units/reaction. The labeled primer(SEQ ID NO. 3) was used at a concentration of 50 nM and the unlabeledprimer concentration was varied in separate reactions for each set ofHIV RNA dilutions, using concentrations of 25, 37.5, 50 or 62.5 nM. Theprobe, which was labeled as specified above and that ultimatelyhybridizes with the product of the labeled primer prior to detection ofthe resultant hybrid complex, was used at a concentration of 10 nM. Theten-fold dilutions of HIV RNA in a sample volume of 25 μl were added to175 μl containing the above mixtures for a total reaction volume of 0.2ml. The negative control was composed of 50 ng of rRNA/reaction. Allreactions were performed in duplicate.

Reaction mixtures were reverse transcribed and amplified in aPerkin-Elmer 480 Thermal Cycler. Reaction mixtures were first incubatedat 62° C. for 30 minutes to reverse transcribe the RNA, followed by 2minutes at 94° C. PCR amplification was then initiated through atouchdown or step-down protocol to aid in the stringency of the reactionin the early stages of amplification. This utilized 8 cycles as follows:1 cycle at 94° C. for 30 seconds then 70° C. for 80 seconds followed by1 cycle of 94° C. for 30 seconds then 69° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 68° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 67° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 66° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 65° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 64° C. for 80 seconds, followed by1 cycle of 94° C. for 30 seconds then 63° C. for 80 seconds. Furtheramplification was then accomplished with 35 cycles at 94° C. for 30seconds then 62° C. for 80 seconds. After the reaction mixtures werethermal cycled, all duplicates were pooled and mixed by pipetting toeliminate any variation due to cycling. The mixtures were then split anddenatured for 5 minutes at 97° C. Following this, probe oligohybridization was accomplished by lowering the temperature to 15° C. for5 minutes The temperature was then lowered to 4° C. and samples wereheld at 4° C. until detection of reaction products.

Reaction products were detected on the Abbott LCx® system (availablefrom Abbott Laboratories, Abbott Park, Ill.). A suspension ofanti-carbazole antibody coated microparticles at 0.06% solids and ananti-adamantane antibody/alkaline phosphatase conjugate (all of whichare commercially available from Abbott Laboratories, Abbott Park, Ill.)were used in conjunction with the LCx® to capture and detect theamplified product/probe hybrid. The enzyme substrate used wasmethyl-umbelliferyl phosphate (MUP), with the rate of conversion of MUPto MU measured and reported as counts/second/second (c/s/s).

Data from this experiment is presented in Table 1 and shows that whenthe unlabeled primer is present at concentrations below that of thelabeled primer higher signals are achieved at higher targetconcentrations, and plateauing of the signal is avoided. Conversely,when the concentration of the unlabeled primer is higher than, or equalto, the concentration of the labeled primer, a “hook effect” is observedwherein a higher target concentration begins to give a lower signal.

TABLE 1 LCx ® Rate (c/s/s) HIV RNA Unlabeled Primer Concentration(molecules) 25 nM 37.5 nM 50 nM 62.5 nM 0 28.2  26.7  26.5  24.1 10¹30.8  29.0  76.3 107.1 10² 39.2  78.6 190.5 299.7 10³ 156.6  389.9 477.7485.1 10⁵ 1017.1  898.8 591.2 334.5 10⁶ 1156.3  942.2 575.5 298.3(Labeled primer was used at a concentration of 50 nM)

This experiment was also performed using a denaturation time of 15minutes instead of 5 minutes, with equivalent results.

Example 3 Detection of HIV Varying the Labeled Primer Concentration

The same HIV RNA sample dilutions used in Example 2 were reversetranscribed, PCR amplified and detected as in Example 2 except twoseparate preparations (G and A) of unlabeled primer (SEQ ID NO. 2), bothat 50 nM, were used and the concentration of the labeled primer (SEQ IDNO. 3) was varied in separate reactions for each HIV RNA dilution set,using 25, 50 and 75 nM of labeled primer. All reactions were performedin duplicate, with duplicate sets pooled after amplification and probehybridization, to eliminate any variation due to cycling. Detection ofreaction products utilized anti-carbazole antibody coated microparticlesat 0.12% and 0.18% solids, in addition to the 0.06% solids used inExample 2. Results are shown below in Table 2.

TABLE 2 LCx ® Rate (c/s/s) at 0.06% solids Unlabeled Primer G UnlabeledPrimer A HIV RNA Labeled Primer Concentration Labeled PrimerConcentration (Molecules) 25 nM 50 nM 75 nM 25 nM 50 nM 75 nM  0 27.925.8 27.0 26.1 28.3 26.7 10¹ 78.5 77.4 40.2 70.6 59.2 90.6 10² 340.4321.2 173.0 319.1 414.1 401.3 10³ 391.0 553.1 574.0 179.7 561.1 750.110⁵ 108.9 860.3 1035.5 34.1 442.7 1130.5 10⁶ 47.4 903.8 1068.6 34.0383.3 1139.8 LCx ® Rate (c/s/s) at 0.12% solids  0 57.7 53.6 52.0 56.856.5 53.6 10¹ 133.6 146.1 78.7 125.0 118.1 185.0 10² 465.0 498.9 312.5461.1 613.0 615.0 10³ 530.6 803.7 859.1 272.6 773.0 1008.3 10⁵ 188.5999.4 1272.7 69.9 581.4 1307.9 10⁶ 90.3 1053.4 1289.2 67.0 512.9 1322.8LCx ® Rate (c/s/s) at 0.18% solids  0 80.9 76.0 77.4 82.5 78.5 77.1 10¹156.9 176.9 101.4 147.3 139.6 226.0 10² 505.2 538.1 374.2 502.9 663.5678.3 10³ 568.5 830.6 911.7 326.9 830.5 1097.4 10⁵ 237.2 1036.1 1296.598.9 617.2 1317.7 10⁶ 117.9 1019.3 1321.6 93.4 544.4 1347.6 (Unlabeledprimers G and A were used at a concentration of 50 nM)

The two unlabeled primer preparations (G and A) resulted in slightlydifferent values but showed the same overall trends. In all cases, whenthe concentration of the unlabeled primer was higher than, or equal to,the concentration of the labeled primer, a “hook effect” was again seen,as in Example 2, wherein a high target concentration gave a low signal.Only when the unlabeled primer was present at concentrations lower thanthe labeled primer was a more linear signal produced correlating withtarget concentration. Under these conditions, as in Example 2, highersignals were again achieved at higher target concentrations, andplateauing of the signal was avoided.

Use of microparticle concentrations of 0.12% and 0.18% solids gaveequivalent results, with both resulting in slightly higher signals thanwhen 0.06% solids was used. However, changing the microparticleconcentration did not affect the overall trends seen due to the ratio ofunlabeled to labeled primer.

Example 4 Detection of HIV at Various Primer and Probe Concentrations

The same HIV RNA sample dilutions used in Example 2 were reversetranscribed, PCR amplified and detected as in Example 3 except theconcentrations of both primers and probe were varied. The unlabeledprimer (SEQ ID NO. 2) was used at concentrations between 100 and 250 nM,varying in 50 nM increments. The labeled primer (SEQ ID NO. 3) was usedat concentrations between 50 and 500 nM, varying in 50 to 100 nMincrements. The probe was used at concentrations of 10, 25 and 40 nM,with the various primer concentrations as indicated in Table 3.

TABLE 3 LCx ® Rate (c/s/s) at 0.12% solids HIV RNA unlabeled primer(nM)/labeled primer (nM)/probe (nM) (mol) 100/50/10 100/100/10100/150/10 100/200/10 150/100/10 150/150/10 150/200/10 150/300/10  048.2 50.3 46.1 44.0 48.2 44.8 46.1 45.0 10¹ 195.7 168.3 140.5 138.7276.8 263.3 216.8 295.5 10² 551.8 599.3 606.5 578.3 594.6 611.1 795.0569.1 10³ 314.6 830.1 1074.2 988.0 828.8 881.9 900.7 865.8 10⁵ 67.7970.9 1200.7 1187.9 188.0 1061.0 1190.0 1020.2 10⁶ 53.8 945.8 1214.11128.2 85.9 1021.8 1173.1 982.7 200/150/10 200/200/10 200/250/10200/400/10 250/200/10 250/250/10 250/300/10 250/500/10  0 42.2 44.8 43.640.9 45.2 44.9 48.1 39.3 10¹ 377.1 192.4 136.0 163.1 111.7 262.3 149.6181.4 10² 569.3 710.7 620.1 486.1 655.2 558.8 558.8 600.5 10³ 593.7850.2 848.7 779.7 797.7 808.3 844.9 820.4 10⁵ 302.1 958.0 1080.3 888.8734.0 963.9 1072.8 930.0 10⁶ 198.6 1050.5 1161.3 913.8 463.8 1018.21174.2 944.9 100/150/25 100/200/25 150/200/25 150/300/25 200/250/25200/400/25 250/300/25 250/500/25  0 47.6 47.8 47.9 46.3 44.7 45.7 44.846.0 10¹ 211.1 239.0 244.3 209.9 184.4 204.2 189.4 92.2 10² 929.0 618.5484.3 638.0 713.5 643.2 1020.1 608.3 10³ 1175.3 1206.0 1049.0 1167.7977.8 1045.3 992.4 1015.4 10⁵ 1619.4 1575.2 1507.0 1392.9 1320.6 1162.91204.6 1106.8 10⁶ 1620.8 1610.2 1551.3 1328.6 1412.3 1194.0 1362.41152.4 100/150/40 100/200/40 150/200/40 150/300/40 200/250/40 200/400/40250/300/40 250/500/40  0 53.9 51.9 52.5 49.3 101.1 51.0 46.3 46.1 10¹544.6 162.8 229.1 273.1 261.7 466.1 167.9 615.0 10² 659.5 759.0 776.8649.8 689.9 565.1 720.9 584.1 10³ 1270.0 1210.2 1189.5 1121.8 978.41046.9 1061.4 1057.1 10⁵ 1824.8 1783.8 1666.5 1612.0 1486.8 1402.61311.3 1368.1 10⁶ 1821.9 1751.6 1742.1 1544.9 1589.3 1397.1 1477.31419.6

All 3 microparticle concentrations were tested but only the data using0.12% solids is shown above in Table 3 since this data wasrepresentative (as in Example 3) of that obtained with all 3microparticle concentrations.

As shown by this example, when the concentration of the unlabeled primerwas higher than, or equal to, the concentration of the labeled primer, a“hook effect” was observed. When the labeled primer was present athigher concentrations than the unlabeled primer the hook effectdissipated.

While the invention has been described in detail and with reference tospecific embodiments, it will be apparent to one skilled in the art thatvarious changes and modifications may be made to such embodimentswithout departing from the spirit and scope of the invention.

1. A method for amplifying and detecting a target nucleic acid in a testsample comprising the steps of: (a) forming an amplification mixturecomprising a test sample, a first primer sequence and a second primersequence, and amplifying agents, (b) amplifying the target sequence togenerate copies of the target sequence comprising an amplificationproduct from the first primer sequence and the second primer sequence,and (c) detecting the copies of the target sequence as an indication ofthe presence of the target nucleic acid sequence in the test sample,wherein the first primer sequence is present at a concentration between100 nM and 250 nM, and the second primer sequence is present at aconcentration of between 50 nM and 500 nM, and wherein the concentrationof the first primer sequence and the second primer sequence differ by atleast 15% and differ by no more than 250%, and wherein the presence ofthe target nucleic acid is indicated by the presence of an amplificationproduct and amplification and detection are efficiently increased byavoiding plateauing of signals.
 2. The method of claim 1 wherein theconcentration of the first primer in the reaction is 20% to 150% greaterthan the concentration of the second primer.
 3. The method of claim 1,wherein the second primer is labeled.
 4. The method of claim 1, whereinthe presence of the target nucleic acid is indicated by contacting thesample with a labeled probe specific for the target nucleic acid.